Oxygenation of steroids by mucorales fungi



y & 1952 H. c. MURRAY ET AL 2,602,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet l M Q g m 92 E u g 5 m0 x O o ?g 5 9 %g 3 D w 11% E! ILL 31: mm D 0 9 U --n W 4 NOISSIWSNVHJ. .LINHQHEM HERBERT fiMURRAY NV o DUREY H.PETERSON 1 EN RS ATTOR NE Y 152 H. c. MURRAY ET AL OXYGENATION OF STEROIDS BY MUCORALES FUNGI 20 Sheets-Sheet 2 Filed Feb. 23, 1952 oovm 000m 00mm NOISSIWSNVHJL HERBERT 0. MURRAY DUREY H. PETERSON INVENTORS ATTORNEY y 1952 H. c. MURRAY ET AL 02,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet 5 CNIPD S BIOCONVERSION BY I7'N-HYDROXYPROGESTER- RHWC ONE CONVERSION SICHRS. COTROLS S48HRS HOP-I6 CONTROLS+ SZR HRS. HRS. HOP-2 1 OP Ig-EOR-24 l66HCM-2l4 a 349-SHE-36 ET SEQ.

PROPYLENEGLYCOL-TOLUENE HOP-l6=l7-a-HYDROXYPRO- GESTERONE (300 I6 HRS FERMENTATION HOP-24=D|TTO,24 HRS. FER- MENTATION CONTROLS:

Il-(l-HYDROXYPROGESTER ONE (GA.24O )DIHY- DROXYPROGE TERONE, DHP-95.4(607) 266-HCM-2l3 8a 349'SHE'32 PROPYLENEGLYCOL- TOLUENE S=COMPOUND S FERMENTATION (320 SUBSCRIPT= HOURS OF IFERMENTNTIO CONTROLS:

COPOUND E (new COPOUND E ("CW COMPOUND s (32% CORTHCOSTERONE (32y) E7616 FIE] HERBERT C. MURRAY NVENTO DUREY H. PETERSON I RS ATTORN E Y Y & H952 H. c. MURRAY ET AL fi fifi OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet 4 u KETOPROGESTERONE- CIWPD s BIOGONVERSION BY IDENTMTY RH I76 H CONTROL 55 CONTROL 5% GON1S98LS+W 266-HCM-2l3 8| 349-SHE-32 A PROPYLENEGLYCOL-TOLUENE m-DHPI s4a=4a HR. FERMENTA'HOM 326-MIU-l26 m-w-uoawc o ommggfgd'flfi fi g g WOW) OFPMPD umwoy) commowwawwoumo HUG-y) CONTRQL- AUTHENTIC; COMPOUND EH6?) SAMPLE ll-KETOPROGES- CQMPQUMD swa TERONE (@Oy) PM 5 WWW HERBERT QMURMY V N DUREY wpmmsow IN E ms y 1952 H. c. MURRAY ET AL 2,602,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet 5 HERBERT C. MURRAY DUREY PETERSON INVENTORS ATTORN E Y July 8, 195

H. c. MURRAY ET AL 5W1? OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 25, 1952 20 Sheets-Sheet 6 0 GHRQATOGIRAM 22 0 (HOP-QM m (HOP-ZM s ERcTaos E FRAGTBONS w w I Bm-DHP-m sna-nHP-uaa MQ-SHE- w 39-SHE-37Q3fl ILL FRACTIONS PPILIED 1r ALL FRACTIONS EEPMED 'n' m LEVEL new LEVEL MMED GOTOLZ MuxEn common;

COMPOUND E My) cowoumo E y DIHYDRUYPGESTERONE HHYDROXYPROGESTEONIE (DHP 95.4) wow (DHIP 95.M(30y) wcnm ouwn E common. wcompouwm momma.

m (lfiy) THE M203 mmomem THE I 0E um E10,, 0F (W0 PROGESTEOME) PROGESTEROME) INVENTOR$ HEBERT EURRM M New IDUREY H. PETERSON y 195 H. c. MURRAY ET AL 02,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 25, 1952 20 Sheets-Sheet '7 PROGESTEROE BHOGOM- PROGESTERONE BIOCON- VERSION OCI 0C2 FROG.

266'HCM-l99/I5OT 549 -SHE-40 (P4) PROPYLENEGLYGOL-TQLUENE IP4=PROGESTERONE EERMEN- mwom (wow 266-HCM-l99-OGI AND 062 363-MIU-2l CARBITOL- METHYLGYCLO- HEXANE CONTROLS:

IDIHYDROXYPROGESTERONE (DHP 95.4 60

Il-fl-HO,PROG STERONE (6.24%)

PROGESTERONE FER- 002 MENTATIONS (500y) PROGF PROGESTERONE con- TROL (80y) HERBERT c. DURIEY H. PETERSON IN V EN m5 Y 1952 H. c. MURRAY ET AL 2&02/769 OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet 8 PROGESTERONE BIOCON- DOC, PROGESTERONE GON- VERSION VERSION CONTROL CONTROL 204DR CORT PROS 6111 Pi P2 DOC 20F 2ss-HcM-2o4oR AND 205PP aes-mu-zz CARBlTAL-METHYLCYCLO- HEXANE 204DR=||-DESOXYGORTICOS- TERONE FERMENTATION (3207) DOG=II-DESOXYCORTICOS- i %%%%1 TIERONE common. (M 39-SHE-4PI M P2 J 9 Q% PROPYLENEGLYGOL-TOLUENE Y) PROG.- PROGESTERONE "mom Home/300m v (300W BOSPP-PROGESTERONE P2=PROGESTERONE FEREN- TATION (HOM'I99/3OQT) FERMENTATION (320y) (300y) g fi lao PROGESTERONE Fla. w

mammompmamm- 0E (DIHIP @5410 HERBERT MURRAY INVENTORS DUREY H. PETERSO FIG. M

July 8, 1952 c MURRAY ET AL 2,602,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet 9 lT-Q-HCLPROGESTERONE GMPD S-ACIETATE BIGCON" BIOCONVERSION VERSION CONTROLS-bull CONTROLS+$ HP HIP EOQ-SA ZOQ'SA 349 SHE-22 PROPYLENE GLYCQL- 1 HEH'WQF- Ho PRQGES- 266-HCM-209SA a349-sHE TERONE Mmwm- PROPYLENE GLYGQL-TOLUENE TION (320;!) 1 ZQS'SIFGOMPOUND S'-AQE- QQNTRQLS; TATE FERMENITATIION DIHYDRQMPRQGESTER- 1 ONE (DHP 95 4)( $0 y) CONTROLS: GOMPOUND Fflfiy) mam H Eflfiy) 8(64y) ATTOR NE Y July 8, 11952 H. C- MURRAY ET AL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 DOC, DOCA BIOCONVERSION CONTROLS+ 02 M2 02 0 1' z'ss-ncm-lse-ozwAz *349-SHE-2I PROPYLENEGLYCOL-TOLUENE D2=ll-DESOXYCORTICOSTER om: (320w omnnmasoxvoommosmn- ONE Acam'rawzoy) CONTROLS D2 CONTROLS 0A2 CONTROLS COMPOUND F 06y) Eflfi Ila 8(6 CORTIGOSTERONE (64y) WWW. l5

HERBERT C. MURRAY DUREY H. PETERSON 2O Sheets-Sheet l0 COMPOUND S BIOCONVERSIOM BY RHW CONTROLS+1 ZGG'HCM-IQS 52 AND 5A2 349SHE' 25 PROPYLENEGLYCOL-TQLUENE S2=COMPOUND S [FERMENTA- WON (320w) 5A2 COMPOUND $"AGETATE FERMENTATHON (320V) CQNTRQLSI COMPOUND F 6y IN V EN TORS ATTOR NE Y July 1952 H. C. MURRAY ET AL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 S @m S-ACETATE BIOGON- B VER S 0 M {CONTROLS-W 1 M SA 5% S Sfl 266-HCM-l9&-S4 AND 8A4 349-8HE-26 PROPYLENEGLYCOL-TOLUENE s COMPOUND s FERMEN- whom 320w s =coMPoumn S-ACETATE FERMENTMIOM (320w commons: COMPOUND FUGy) COMPOUND E06) GOWWPQUND 20 Sheets-Sheet ll CMPD S BIOCONVERSBUN BY M I GOMTWULS 349-SHE-48 PROPYLENEGLYCOL TOLUENE I=COMPOUND s FERMEN- TATION (320 CONTROLS:COMPOUND EH67) compoumo Eflfiy) COMPOUND swa FIE. IQ

HERBERT C. MURRAY INVENTORS DUREY IHI. PETERSON ATTORN E Y y 1952 H. c. MURRAY ET AL 02,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet l2 ANDROSTENEDIONE BIOCON- ANDROSTENEDIONE BIOCON- VERSION VERSIO AND AR DOC PROG 8 AND AR DOG FROG 5 T W w,. Q u

. e-ucm- 2| mama zee-ucm-zlsARa 4 -SH 70.3 PROPYLENEGLYGOL-TOLUENE GARBITOL-METHYILGYGILO- AND=ANDROSTENEDIONIE com- HEXANE TROI- v) mmmmosmmemom AR= ANDIROSTENEDHONE F|ER- CONTROL (80y) I MENTATION (500w AR=ANDROSTENEDIONE FER- 1000 u-masoxmomncos- MENTATION (50W) TERONE CONTROL (64y) DOG =||-0EsoxYcomIos- PROG=PROGESTERONE COM- TERONE GONTROUG) TROL (80y) PROG=PROGESTERONE coms =COMPOUND s common. TROL (BQW (32y) smowoumn 5 common.

(32y) H620 F1612! HERBIET EU Y INVENTORS DUREY H. PETES ATTORN E Y y 9 1952 H. c. MURRAY ET AL 9 3 OXYGENATION OF STEROIDS BY MUCORAL-ES FUNGI Filed Feb. 25, 1952 20 Sheets-Sheet 1s CHOLESTENONE BIOGOM- ERGOSTEROME BIOGOM- VERSION! VERSIQM Y GHOL DOG E D06 266 HOM-2l9CR AND 349- SHE-70.2 CARBITOL-METHYLCYCLO- HEXANE CHOL=CHOLESTENONE cou- TROL (80y) CR= OHOLESTENONE FER- MENTATION (5007) DOC= a uossoxvcoRTlcos- TERONE CONTROL (64 PROG=PROGESTERONE cou- TROL (64y) 266'HCM'2I9'OR AND 549- SHE-70.l

GARBITQL-METHYLCYCLO- HEXANE OR=ERGOSTERONE FERMENI- TATION (500)) ERG=ERGOSTERONE CON- ROL (8O FIGHZE FWZ HERBERT c. URRY INVENTORS DUREY H. PETERSON ATTORNE Y Jufly 8, 1952 H. C. MURRAY ET AL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 ERGO STERQL S EON a1 1;IOGONVER CONTROLS E8 E8 @ONTROLS ER=ERGOSTEROL FERMEN- TATBON (SQOW CONTROLS! H-DESOXYCORTIICOSTERONE ERGOSTEROIL.

GOMPOUND 2O Sheets-Sheet l4 PROGESTERONE CONVERSION VEGETATWE HNIOGUILATIOM SHMPILE MEDIIUMI CON TRULS VEG. +VE55. CONT 3 2 C -22OV&549-SHE- 53.2 PROPYLENEGLYCOL-TOLUENE VEG=PROGESTERONE FER- MENTATION CONTROLS: II- a- HQ, PROGESTEIRONE DIHYDROXYPRQGESTERONE HERBERT 0. MURRAY H. ETERSQ JNVENTORS ATTORNEY July 8, T5

H. C. MURRAY ET AL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 25, 1952 PROGESTERONE BIOCON- VERSION BY M CONTROLS 3|4AW-64-7I 8: 349- SHE-47 PROPYLENEGLYCOL-TOLUENE Ml=MI/P6 PROGESTERONE IFERMENTATION (3207) M4=M4IP6 PROGESTERONE FERMENTATION (3207) M5=M5IP6 PROGESTERONE FERMENTATIOR (320w) CONTROLS:

lfl--HO PROGESTERONE 1 DIHYDROXYPROGES- TE ONE (DRP 95.)(30y) 2O Sheets-Sheet l5 PROGESTERONE BIOCON- VERSION BY MUCORALES CONTROLS 2 M6 A 3I4-AW-64-7l a 349-sHE-4o PROPYLENEGLYCOL-TOLUENE M2=M2IP6 FERMENTATION (mo M3=M3IP6 FERMENTATHON (240 M6=M6IP6 FERMENTATION (240 CONTROLS:

llQ-HO PROGESTERONE (3o OIHYDROXYPROGES- TEOE (DHP 95mm B-HQ-HOWPROGESTEONE HERBERT G. MURRY INVENTORS DUEY H. PETESO ATTORNEY July 8, 1952 H. C. MURRAY ET AL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 PROGESTERONE BlOCONVER- SIOM BY MUCORALES CONTROLS PROPYLENEGLYCQL-TOLUENE M7= MT/PG FERMENTATUON M8= M8/P6 FE RMENTATIONI GONTROLSI ll-Cl-HO, PROGESTERONE DIHYDRQXYPRQGESTIERONE FIG.

2O Sheets-Sheet l6 PROGESTERONE BIOCONVER- SION BY MUGORALES CONTROLS PROPYLENEGLYCOL 'TOLUENE Rl= Rl/PG FERMENTATION R2= R2/P6 FERMENTATION R6= R6/P6 FERMENTATION CONTROLS:

Ila- H0 PROGESTERONE DIHYDROXYPROGESTERONE (DHP 95mm,

HERBERT QMURRAY INVENTORS DUREY H. PETERSON ATTORNEY July , H. c. MURRAY ETAL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 25, 1952 PROGESTERONE BIOCONVER- snow BY MUGORALES omma.

3l4-AW-64-7I a 349-SHE-4O PROPYLENEGLYCOL-TOLUENE 3= m/Pe FERMEMTMION (207) R4=R 7P6 FERENTATIOM CONTROLS;

q na--Ho,, PROGESTER- wow mwmmxv- POGIESTERQME (0MP (30y) Pm-H0 PRUGIESTER- ONEWOW FIG. 30

2O Sheets-Sheet l7 PROGESTERONE BIOCONVER- SION BY UCORALES 5 7 R HONTROLS PROPYLENEGLYCOL-TOLUENE R?gg/6 FERMENTATION RT= R7/ P6 FERENTATION R8=R8IP6 FERMENTATION GONTROLSIlfl-fi- HO PRO- GESTERONE a s4 ("30 DHHYDROYPRO WP INVENTORS FIE; .W ERET G. FY 1 3?; MI. ETERSQN ATTORNEY H. C. MURRAY ETAL OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 DOG BflOOONVERSION BY NUGORALES CONTROLS Nil N2 3 earvr- O O 3|4-AW-64-7l & 349-SHE-50 PROPYLENEGLYOOL-TOLUENE NH=NIIDOG6 FERETATION (320w) 2=N2IDOC6 FE EN'II'THO (320W N3=3IDOC6 FERNIENTTION CONTROLS: NNXTURE OIF COMPOUND F (B6)) COMPOUND IE (l6 OOTflCOSTEON (6%!) 2O Sheets-Sheet 18 ICONVERSION BY 3|4-AW-64-7l 8x 349'SHE-5O PROPYLENEGLYCOL-TOLUENE 2=2/DOC6 FERMENTATIO (320w CONTROLS: OF

COPOUND F (I67) GOPOUND E (I67) OORTIOOSTERONE (64 HERBERT C. MURRAY NVENT R DUNEY H. PETERSON I 0 S ATTOR NE Y Y W52 H. c. MURRAY ETAL 2,602,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet l9 PROGESTERONE GONVER- SIGN BY BEER FIILTRATE GOM'II'ROL. 82 H1 349'SHE-4l GONTROL= PROGESTERONE SI=FERMENTATION BY DI- SH 5 LUTED BEER FILTRATE PRQPYLENEGLYGOL-TOLUENE PH 6.76 MH=MIIDOG 6 FERMENTATEOM 32= UKE 5 BUT PH 4 55 (329W R=FERMENTATIOM BY MY- wmams: mum OF GELIUMI WMPWND W BF=FERMENTATBON BY UN- GOMPOUNU E (E6 D DILUTED BEER FILTRATE GUR'HGUSTERQJ (64y) PH 4.!

CARBH'II'QL-METHYLGYGLO- H6. HEMNE DUREY M. PETERSON ATTORNE Y July 8, 1952 H. c. MURRAY ET AL 2,602,769

OXYGENATION OF STEROIDS BY MUCORALES FUNGI Filed Feb. 23, 1952 20 Sheets-Sheet 20 ll--@- H0 PROGESTERONE CON- VERSION BY RH I76 CONTROLS 72 +72 CONTROLS k HGM- 8m 9'SHE-72 PROPYLENEGLYGOL-TOLUENE 72= film-H0, PROGESTERONE FERENTATIQN (320W GOTROLSI H-HO PROGESTERONE (60y) DBHYDROXYIPROGESTERONE (DHP 95M 30y) FIG. 36

HERBERT c. MURRAY ENTO DUREY PETERSON my R5 ATTORN E Y 

1. A PROCESS FOR THE PRODUCTION OF AN ELEVEN OXYGENATED STEROID COMPRISING THE STEP OF AEROBICALLY SUBJECTING A STEROID CONTAINING AN ELEVEN METHYLENE GROUP TO THE ACTION OF OXYGENATING ENZYMES PRODUCED BY A GROWTH OF AN OXYGENATING SPECIES OF FUNGUS OF THE ORDER MUCORALES. 